1. Field of the Invention
This invention pertains generally to compositions and treatments for controlling unregulated cellular proliferation in mammals, and more particularly to the preparation and use of Tachykinins, preferably Sialokinins, to modulate unwanted cellular proliferation.
2. Description of Related Art
Studies from the recent past indicate that the immune system and the nervous system are integrated forming an interdependent neuroimmune network rather than existing as independent systems. This network includes a complex array of endocrine, neurocrine, autocrine and paracrine interactions between different cell types within the two systems. The reciprocal flow of information between the nervous and immune systems uses an extensive array of mechanisms usually involving soluble signaling molecules or cell to cell contacts. The network also includes positive and negative signals as well as feed forward and feedback loops that control neuroimmune functions.
The coordination, regulation and amplification of the mammalian neuroimmune network pathways involve many different classes of molecules including neurohormones, cytokines, non-peptide mediators and neurotransmitters such as tachykinins, enkephalins and endothelins. For example, significant concentrations of neuropeptides produced by nerve cells are found at the sites of inflammation and immune reactions acting on receptors expressed on immunocytes and may be involved in the eradication of infected cells as well as modulating the proliferation of cells for repair.
Likewise, pro-inflammatory cytokines that may be released from activated macrophages or monocytes have been shown to target nervous system cells that control sleep, mood and temperature during infection.
Cytokines are a diverse group of immunomodulatory proteins that can modulate the biological activity and proliferation of cells and tissues. Unlike hormones that are produced by specialized cells or glands, cytokines are secreted proteins produced by a number of cell types in different parts of the body. Cytokines have biological activity that may be similar to some hormones but have a broader spectrum of target cells than hormones. Furthermore, almost all of the cytokines are pleiotropic in nature exhibiting multiple biological activities. Cytokines include Interlukins, Lymphokines, Monokines, Interferons, colony stimulating factors and Chemokines.
Cytokine receptors are found on the cells of both the nervous and immune systems. Molecular signals can induce or repress the synthesis of cytokines and cytokine receptors in the same cell or sequentially on different cells. Some cytokine receptor proteins have been shown to function as signal transducers within the cell as well as binding the cytokine ligand due to the tyrosine kinase activity of the receptor. It can be seen that the cytokines are important positive and negative regulators of cell survival and death, mitosis, cell transformation and cell differentiation as well as the systemic responses to infection such as inflammation.
Some viral strategies exploit the activity of various cytokines in order to evade an immune response from the host. For example, virus encoded cytokine analogs may be produced that antagonize or agonize the host cytokine receptors. The virus may produce a protein that inhibits the synthesis and release of cytokines from infected cells or interferes with the interaction between the host cytokines and their receptors to avoid destruction of the infected cell through apoptosis.
There are also a number of low molecular weight neuropeptides that display many of the same properties as cytokines but are not normally classified as cytokines or growth factors because of their size. Some of these low molecular weight proteins and peptides include Vasopressin, Bombesin from amphibians, Bradykinin and the Tachykinins. Many of these neuropeptides are pleiotropic and have dose and time dependent effects that provide a functional redundancy to cytokines.
The Tachykinins are a family of neuropeptides that were initially identified as neurotransmitters. Although they are not classified as cytokines, tachykinins also exhibit cytokine like activity. For example, tachykinins can activate neutrophilic granulocytes and stimulate the proliferation of T-cells. Tachykinins may also be involved in the release of cytokines by macrophages and granulocytes such as Interleukin 1 (IL1), Interleukin 6 (IL6), and (TNF-alpha) (IFN-gamma). Tachykinins may also stimulate the secretion of immunoglobulins as well as be involved in the regulation of non-infection inflammatory responses.
The tachykinin family of neuropeptides includes Substance P (SP) SEQ. ID. NO. 3; Neurokinin A (NKA) SEQ. ID. NO. 4; Neurokinin B (NKB) SEQ. ID. NO. 5; Neuropeptide K (NPK) SEQ. ID. NO. 6 and Neuropeptide gamma (NPγ) SEQ. ID. NO. 7. Each member of the family has a conserved amino terminal sequence ( . . . Phe-X-Gly-Leu-Met-NH2) SEQ. ID. NO. 8. With the exception of NKB, all of the neuropeptides are encoded by the prepro-tachykinin-I gene (PPTI). The PPTII gene encodes Neurokinin B. The sequence of the tachykinin neuropeptides can be seen in Table 1.
Substance P was first identified in 1931 from brain and gut extracts that exhibited smooth muscle contractile properties. The amino acid sequence of Substance P was first identified in 1971. Subsequent studies of the pharmacological properties of SP, NKA and NKB led to the identification of three distinct neurokinin receptors with preferred ligands. Substance P preferentially binds to the NK1 receptor. NKA and NKB preferentially bind the NK2 and NK1 receptors respectively. However, each type of neurokinin is not highly selective and may bind to all receptor types with various affinities. In addition, these receptors may also bind Bombesin and similar peptides such as gastrin-releasing hormone under certain conditions.
The tachykinin receptors NK1, NK2 and NK3 are from a group of transmembrane-spanning G protein-coupled receptors and have been associated with the activation of different second messenger systems within the cell. It is part of a system that converts external signals into intracellular messages. Generally, the receptors have a transmembrane domain that passes through the membrane seven times that has a highly conserved sequence. The amino terminal section of the receptor is positioned on the exterior of the cell and interacts with the ligand and the carboxyl terminal section is on the interior of the cell and is associated with one or more G-proteins. G-proteins typically have three subunits that can associate with a receptor, a target molecule and GDP or GTP. The G-protein can initiate cellular events directly or through a cascade of signaling events. For example, Substance P can stimulate phospholipase C and phospholipase A2 activity in different cell systems as a result of the activation of tachykinin receptors. Tachykinin receptor activation may also modulate calcium or sodium ion channel function in the cell.
It has been seen that Substance P (SP) and NKA are released from airway sensory nerves upon exposure to irritant chemicals and endogenous agents including bradykinins, prostoglandins, histamine, and protons. The released neuropeptides are potent inducers of a cascade of responses, including vasodilation, mucus secretion, plasma protein extravasation, leukocyte adhesion-activation, and bronchoconstriction. NK1 receptors that are preferentially activated by SP are important for inflammatory actions, while NK2 receptors that are preferentially activated by NKA mediate bronchoconstriction. Disease states such as inflammation or viral infections lead to enhanced peptide synthesis and an increased sensory nerve excitability. Together with increased NK1 receptor synthesis and loss of major tachykinin-degrading enzymes such as neutral endopeptidase in airway inflammation, it has been suggested that recently developed, orally active nonpeptide neurokinin receptor antagonists could have a therapeutic potential in asthmatic patients. The released sensory neuropeptides are potent inducers of a cascade of responses collectively called neurogenic inflammation that have a similarity to several features of asthma.
Tachykinins also have been shown to increase vascular permeability in rat and guinea pig airways through the opening of endothelial gaps at postcapillary venules; SP is more active than NKA in this respect. Studies using receptor-selective synthetic agonists have suggested the involvement of NK1 receptors for this effect. Tachykinins also potently increase airway blood flow, including trachea and nasal and bronchial mucosa, presumably via NK1 receptors. Tachykinins exert bronchoconstrictor effects in most species, including man. NKA is more potent than SP in this respect. The NK2 receptor mechanism has a revealed dominance for this response in human and guinea-pig bronchi. In the guinea pig, NK1 receptors also mediate tachykinin contraction of bronchial smooth muscle, although it is less likely that tachykinins released from sensory nerves activate these receptors.
Substance P has also been shown to be a potent stimulator of airway mucus secretion in man and SP is a likely mediator of increased goblet cell discharge after exposure to cigarette smoke. By using selective agonists and antagonists the involvement of NK1 receptors in tachykinin-induced mucus secretion from the airways has recently been defined. The potent secretagogue properties of SP on mucus have been confirmed in human nasal mucosa. Tachykinins also potentiate cholinergic neurotransmission in lower airways, mainly via NK1 receptors. Tachykinins can modulate the function of a variety of inflammatory cells. Some of these reactions, e.g., mast cell and eosinophil granulocyte degranulation, are likely to be unrelated to the stimulation of specific neurokinin's and probably caused by direct stimulation of G proteins. There is no evidence that tachykinins degranulate lung mast cells in contrast to the situation in the skin. Substance P in very low concentrations may act as a mast cell primer to other agents when released from sensory nerves rather than as a direct degranulating agent. Alveolar macrophages are activated via NK2 receptor stimulation. Furthermore, neutrophils are recruited upon sensory nerve activation in rat airways.
Tachykinins may also be involved in more long term effects, since both SP and NKA stimulate chemotaxis and proliferation of human lung fibroblasts, indicating a role in lung fibrosis. It is possible that both NK1 and NK2 receptors are involved in this response. Exposure to irritants causes sensory mechanisms to be upregulated. An increase in the responsiveness to tachykinins and an increase in the number of postcapillary venules contribute to the augmented plasma extravasation in these conditions. The number and length of SP-containing nerves in lower airways are increased in patients with fatal asthma. There is an increased expression of NK1 receptors observed in the lungs of asthmatics and the reactivity to tachykinins is greater in allergic subjects both regarding bronchoconstriction and nasal congestion as well as wheal and erythema in the skin. (See Lundberg, Jan M. (1995) Tachykinins, sensory nerves, and asthma—an overview. Can. J. Physiol. Pharmacol. 73: 908-914).
Neurotransmitter receptors have also been shown to have the capacity to act as regulators of cellular proliferation, including airway smooth muscle (ASM) cells. Chronic asthma is characterized by ASM hyperplasia. SP induces a potent dose-dependent stimulation of ASM cell growth. However, NKA and NKB demonstrated little or no appreciable effect on airway smooth muscle cell proliferation.
These neuropeptides induce airway smooth muscle cell mitogenesis via transmembrane signaling mechanisms associated with specific activation of the NK1 receptor. SP induced dose-dependent increases in ASM cell number within the concentration range of 10−14 to 10−4M. The maximum proliferative response was elicited with 10−4M. Neither NKA nor NKB induced significant proliferative responses in the ASM cells within the dose range of 10−12 to 10−6M, with the exception of NKA eliciting a relatively modest increase in ASM cell count at the highest administered concentration of 10−4M. Thus, tachykinin-induced ASM cell proliferation is mediated by selective activation of the NK1 receptor.
Activation of the NK1 receptor is also coupled to the activation of phospholipase A2 and phospholipase C via a PT-insensitive mechanism. Tachykinin-induced ASM cell growth was largely mediated by activation of NK1 receptors. The pro-mitogenic effect of SP in cultured human skin fibroblasts is largely mediated by its interaction with the NK1-receptor subtype. However, NK2 and NK3 receptor activation failed to show an inhibitory action on ASM cell growth. SP can elicit stimulation of phospholipase C and phospholipase A2 activity in different cell systems. ASM cells were found to be coupled to activation of phospholipase A2, the latter resulting in the release and pro-mitogenic autocrine action of certain eicosanoids, including thromboxane A2 and leukotriene D4. The pro-mitogenic action of SP is inhibited by selective blockade of the NK1 receptor. The proliferative response to SP is near half-maximally blocked either by inhibition of phospholipase C or of phospholipase A2. (Noveral, James P., and Michael M. Grunstein. (1995) Tachykinin regulation of airway smooth muscle proliferation. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L339-L343).
Accordingly, there appears to be SP-induced regulation of airway smooth muscle cell growth and that the action of SP is mediated by transmembrane signaling events coupled to selective activation of the NK1 receptor. The latter, together with recent evidence that NK1 receptor gene expression may be enhanced in the lungs of asthmatic patients suggests a potentially important role for tachykinins, principally SP, in the pathogenesis of the airway smooth muscle remodeling found in asthma.
The neuropeptide Substance P was also found to stimulate DNA synthesis and cell growth for epithelial cells (cornea and lens) in a serum-free environment. Recently, it was also reported that SP stimulates release of PGE2 and proliferation in rheumatoid synoviocytes. These findings are in accord with other evidence that indirectly suggests that the release of tachykinins from sensory nerves in the skin, joints, and other peripheral tissues might function as mediators of local inflammatory and wound healing responses. In short term studies, (40 h) it was found that for lens and cornea epithelial cells, SP could stimulate DNA synthesis in a serum free environment; however, the lens cells were less responsive to SP unless insulin was present, while the cornea epithelial cells were sensitive to SP alone but showed little synergism in the presence of insulin. The results of pre-treating cells with SP, followed by the addition of either insulin or more SP, showed that addition of SP for a short time (2 h) can have an effect on the stimulation caused by subsequent addition of a second hormone. This would seem to indicate that either the dissociation rate for SP from its receptor is slow, or that the internal signals for DNA synthesis persist for quite some time.
Additionally, Substance P has been shown to activate malignant glial cells to induce cytokine release and proliferation, both responses being relevant for tumor progression. In various astrocytic/glial brain tumor-derived cell lines, the presence of tachykinin NK1 receptor was seen to strictly correlate with the effect of SP or NKA of increasing DNA synthesis and cellular proliferation. In addition, SP may control many other glial responses such as taurine release, secretion of various cytokines (e.g. Interleukin IL-6, IL-8, transforming growth factor-β, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor) that are thought to be relevant for glioma progression. In fact, SP activates phospholipase C and stimulates the release of IL-6 and prostaglandin E2 from human fetal astrocytes in culture, and there is evidence for an up-regulation of this receptor by reactive proliferating astrocytes after transection of the optic nerve.
In addition, SP-immunoreactive astrocytes have been observed in multiple sclerosis plaques and in the forebrains of human infants. In fact, astrocytoma and glioblastoma cells contain significant levels of high affinity receptors for SP and their increase in expression correlate with the most malignant phenotype.
A human ovarian carcinoma cell line, lacking in tachykinin NK1 receptors and unresponsive to SP in terms of cell proliferation and DNA synthesis was used as a proof to assess the specificity of tachykinin NK1 receptor activation in glioma growth. These data suggest an involvement of tachykinins in supporting glioma progression. The presence of SP receptors in glioma cells is correlated with an increase in mitogenesis and cytokine release responsive to SP. The soluble factors (cytokines, prostaglandins, taurine) induced by SP activation in glioma cells are themselves tumor growth factors and can influence the tumor cell-host interactions including depression of the immune response, angiogenesis, and microenvironment modifications.
It has also been observed that the cells lining the mosquito larval gut as well as the saliva gland of the water strider display multiple chromosome copies e.g. endopolyploidy because something appears to inhibit the cell from dividing after the chromosomes have been replicated. The occurrence of endopolyploidy is believed to be the result of tachykinins present in the gut of mosquitoes as well as in the salivary glands of water striders. The saliva of the mosquito Aedes aegypti has been shown to contain a 1400-Da vasodilatory peptide with a sequence and pharmacological properties characteristic of a tachykinin. For example, the salivary peptide has been shown to have similar biological activity to mammalian tachykinins in endothelium-dependent relaxation of aortic ring studies and contraction of guinea pig ileum preparation studies as well as having cross desensitization with the vertebrate tachykinin substance P, and a positive reaction with anti-substance P antibodies.
The acquired mosquito vasodilator was purified to homogeneity and found to consist of two peptides: Sialokinin I SEQ. ID. NO. 1, with the sequence Asn1-Thr2-Gly3-Asp4-Lys5-Phe6-Tyr7-Gly8-Leu9-Met10-NH2, and Sialokinin II, SEQ. ID. NO. 2, identical to Sialokinin I except for the substitution of aspartic acid (Asp) for asparagine (Asn) in position 1. Sialokinin I (SK1) is typically present in amount of 0.62 μmol (711 ng) and Sialokinin II (SK2) is typically present in 0.16 μmol (178 ng) per salivary gland pair.
When assayed on the guinea pig ileum, both peptides had very similar potencies to that shown by substance P in a comparable assay, with K0.5 values of 6.58 nM for the Asn-derivative, 5.07 nM for the Asp-form, as compared to 4.94 nM for Substance P. There is some indication that the mosquito tachykinins produce stronger contractions at concentrations of 3×10−8 M and above, but the physiological relevance of this is uncertain. (See Champagne, D. E. and J. M. C. Ribeiro (1994) Sialokinin I and II: Vasodilatory tachykinins from the yellow fever mosquito Aedes aegypti. Proc. Natl. Acad. Sci. 91, 138-142.)
Later, in a study of carboxyl-terminal heptapeptides having the sequence (I-II-Phe-III-Gly-Leu-Met-NH2), SEQ. ID. NO. 8, it was determined that two classes of tachykinins could be defined. The first class includes those peptides with Gln or Asn in position I and an aromatic amino acid in position III, related to substance P, and those with an aspartic acid (Asp) amino acid in position I and an aliphatic amino acid valine (Val) or isoleucine (IIe) in position III, related to Neurokinin A and Neurokinin B.
Substance P and related compounds interact with the NK1 receptor, present in the guinea pig ileum among other tissues, and neurokinin-like peptides interact with NK2 and NK3 receptors, found for example in rat duodenum. It can be seen that the sialokinins mix characters of both tachykinin types, having an (Asp) amino acid at position I and a tyrosine (Tyr) amino acid at position III.
The model peptide results predict that the Sialokinins should be less active than substance P on the guinea pig ileum, but this was not observed to be the case. Although Munekata et al. concluded that the identity of the amino acid II was not important, the basic character of (Lys) in position II might compensate for the acidic (Asp) in position I, producing a more substance P-like carboxyl-terminal region with higher affinity for NK1 receptors.
It can also be seen that tachykinins produce a variety of effects in addition to smooth muscle contraction and endothelium-dependent dilation. Substance P causes the release of histamine from mast cells and enhances human neutrophil phagocytosis, but these effects appear to be dependent on the sequence of basis of amino acids (Arg-Pro-Lys-Pro), SEQ. ID. NO. 9, at the carboxyl-terminal end and require micromolar concentrations in vitro or 100 nM concentrations in vivo. Some tachykinins that lack this sequence may inhibit the effect, because the carboxyl-terminal sequence is also involved in binding to a receptor on mast cells. On the other hand, substance P and neurokinins can activate macrophages at low concentrations, concentrations comparable to those required for smooth muscle contraction.